SPARC binding peptides and uses thereof

ABSTRACT

The invention provides SPARC and Albumin binding peptides for the targeting of disease sites, such as tumors, with therapeutic and diagnostic agents. In particular, compositions comprising SPARC binding peptide-Antibody Fc domain fusion proteins and methods of their use are disclosed.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.13/132,274, filed on Oct. 11, 2011, the national stage of InternationalPatent Application No. PCT/US2009/066986, filed on Dec. 7, 2009, whichclaims the benefit of U.S. Provisional Application No. 61/120,228, filedon Dec. 5, 2008. The complete contents of which are hereby incorporatedby reference.

This application claims the benefit of U.S. Provisional Application No.61/120,228, filed on Dec. 5, 2008, the complete contents of which ishereby incorporated by reference.

BACKGROUND OF THE INVENTION

Secreted Protein, Acidic, Rich in Cysteines (SPARC), also known asosteonectin, is a 303 amino acid glycoprotein which is expressed in thehuman body. SPARC expression is developmentally regulated, with SPARCbeing predominantly expressed in tissues undergoing remodeling duringnormal development or in response to injury. See, e.g., Lane et al.,FASEB J., 8, 163-173 (1994). For example, high levels of SPARC proteinare expressed in developing bones and teeth, principally osteoblasts,odontoblasts, perichondrial fibroblasts, and differentiatingchondrocytes in murine, bovine, and human embryos. SPARC also playsimportant roles in cell-matrix interactions during tissue remodeling,wound repair, morphogenesis, cellular differentiation, cell migration,and angiogenesis, including where these processes are associated withdisease states. For example, SPARC is expressed in renal interstitialfibrosis, and plays a role in the host response to pulmonary insults,such as bleomycin-induced pulmonary fibrosis.

SPARC is differentially expressed in tumors and its surrounding stromain various cancers in comparison to the normal tissue, with the patterndepending on the type of cancer. Thus, there is no unifying model whichexplains all facets of its function and contribution to the developmentand progression of cancer. In one pattern, increased SPARC expressionhave been reported in breast cancer (Bellahcene and Castronovo, 1995;Jones et al., 2004; Lien et al., 2007; Porter et al., 1995), melanoma(Ledda et al., 1997a), and glioblastomas (Rempel et al., 1998).Increased SPARC expression plays a role in tumor promotion orprogression in these cancers.

Accordingly, SPARC over expression in inflammation and some cancersmakes a SPARC potential target for diagnosis and therapy.

BRIEF SUMMARY OF THE INVENTION

The invention provides compositions for delivering a therapeutic ordiagnostic agent to a disease site in a mammal comprising atherapeutically or diagnostically effective amount of a pharmaceuticalcomposition comprising the therapeutic or diagnostic agent coupled to aSPARC-binding-peptide (“SBP”) and a pharmaceutically acceptable carrier(“inventive compositions”), including wherein the SBP comprises one ormore of SEQ ID NOs: 1-117.

Particularly preferred embodiments include, e.g., inventive compositionsfor delivering a therapeutic agent to a disease site in a mammalcomprising one or more SBPs, wherein the therapeutic agent is anantibody fragment comprising a functional antibody Fc domain, including,e.g., wherein the functional antibody Fc domain comprises SEQ ID NO:118.

Additional preferred embodiments include inventive compositions fordelivering a therapeutic or diagnostic agent to a disease site in amammal composition, e.g., wherein the SBP comprises: at least 10consecutive amino acids from any one or more of SEQ ID NOs: 1-112 and117. Preferably, the SBP can be comprised of at least 10 consecutiveamino acids from any one or more of SEQ ID NOs: 1-112 and 117. Otherembodiments include compositions, e.g., wherein there are two or moreseparate SBPs, wherein each individual SBP comprises at least 10consecutive amino acids from any one of SEQ ID NOs: 1-112 and 117,preferably any one or more of SEQ ID NOs: 1-5. Embodiments includecompositions, e.g., wherein there are two or more separate SBPs, whereinthe individual SBPs are comprised of one or more of SEQ ID NOs: 1-117.

The invention also provides compositions for delivering a therapeutic ordiagnostic agent to a disease site in a mammal comprising atherapeutically or diagnostically effective amount of a pharmaceuticalcomposition comprising the therapeutic or diagnostic agent coupled to aSBP, pharmaceutically acceptable carrier, and a pharmaceuticallyacceptable carrier, further comprising an albumin binding peptide(“ABP”), wherein the ABP comprises a SEQ ID NO: 119 or SEQ ID NO: 120 orboth SEQ ID NOs: 119 and 120. Such compositions include, wherein the SBPand the ABP are in the same polypeptide and wherein the SBP and the ABPare in different polypeptides.

The invention further provides methods for delivering a therapeutic ordiagnostic agent to a disease site in a mammal comprising atherapeutically or diagnostically effective amount of a pharmaceuticalcomposition comprising the therapeutic or diagnostic agent coupled to aSPARC-binding-peptide and a pharmaceutically acceptable carrier,(“inventive methods”) wherein the SBP comprises SEQ ID Nos: 1-117.Preferred embodiments include inventive methods wherein thecompositions, e.g., wherein the SBP comprises: at least 10 consecutiveamino acids from any one or more of SEQ ID NOs: 1-112 and 117, morepreferably from any one or more of SEQ ID NOs: 1-5 and 117.

Other preferred embodiments include inventive methods, e.g., whereinthere are two or more separate SBPs, wherein the individual SBPs arecomprised of one or more of SEQ ID NO: 1-117. The invention alsoprovides inventive methods, wherein there are two or more separatepolypeptides each comprised of at least one SBP and wherein the SBPscomprise at least 10 consecutive amino acids from any one of SEQ ID NOs:1-112.

Particularly preferred inventive methods include compositions, e.g.,wherein the therapeutic agent is an antibody fragment comprising afunctional antibody Fc domain such as wherein the antibody fragmentcomprises SEQ ID NO: 118. Such methods in accordance with the inventioninclude, e.g., wherein the therapeutic agent is an antibody fragmentwhich mediates one or more of complement activation, cell mediatedcytotoxicity, inducing apoptosis, inducing cell death, and opsinization.

The inventive methods provided by the invention also include serumalbumin-binding-peptides (“ABPs”) comprising SEQ ID NOS: 119 or 120 orboth SEQ ID NOS: 119 and 120. Methods in accordance with the inventionfurther include, e.g., both wherein the SBP and the ABP are in the samepolypeptide and wherein the SBP and the ABP are in differentpolypeptides. However, the SBP can also be comprised of at least 10consecutive amino acids from any one or more of SEQ ID NOS: 1-112.

The inventive compositions and inventive methods provided can beemployed wherein the disease site is a tumor and wherein the mammal is ahuman patient.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 depicts the general concept of fusing a binding peptide to atherapeutic or diagnostic agent. In the example depicted in thisdrawing, the therapeutic agent is an antibody Fc domain.

FIG. 2 depicts the general strategy for the iterative screening of aphage display library.

FIG. 3 depicts the sequences identified after screening a peptide phagedisplay library for binding to SPARC by number of times the sequence isisolated.

FIG. 4 depicts the sequences identified after screening a peptide phagedisplay library for binding to SPARC by the avidity of binding to SPARC(as indicated by OD).

FIG. 5 depicts the cloning of either peptide peptides PD 15 or PD21 intothe pFUSE-hIgG1-Fc2 vector to give PD15 or PD21 Fc fusion protein.

FIG. 6 demonstrates the DNA sequence resulting from the cloning of thesequences encoding peptide 15 and peptide 21 into the pFUSE-hIgG1-Fc2vector to encode peptide-Fc fusion proteins.

FIG. 7 depicts the expressed and purified PD 15-Fc and PD 21-Fc fusionproteins in polyacrylamide gel electrophoresis.

FIG. 8 depicts the Protoarray used to define off target binding of PD 15and PD21.

FIG. 9 is a graph of ELISA binding assays comparing the avidity of SPARCbinding by PD 15 and PD 21 to that of an anti-SPARC antibody.

FIG. 10 presents photomicrographs of immunohistolgic studies performedon sections of a human tumor demonstrating tumor SPARC expression withan anti-SPARC antibody (R&D Anti SPARC). The negative controlanti-Herceptin antibody (Fc fragment only) and a Stablin bindingpeptide-Fc fusion protein (stab-Fc) do not stain the tumor.

FIG. 11 depicts the histologic staining of a SPARC expressing tumordemonstrating the binding of PD 15 and PD 21 to the SPARC expressingcells of the tumor.

FIG. 12 depicts a potential SPARC binding site on elastin.

FIG. 13 depicts the antitumor activity of PD 15 and PD 21 in a humanprostate cancer/nude mouse model system.

FIG. 14 depicts the antitumor activity of PD 15 and PD 21 in a humanbreast cancer/nude mouse model system.

FIG. 15 depicts two svFc polypeptides, ScFv 3-1 and ScFv 3-2, with SPARCbinding activity.

FIG. 16 depicts two svFc polypeptides, ScFv 2-1 and ScFv 2-2, with SPARCbinding activity.

DETAILED DESCRIPTION OF THE INVENTION

SBPs and ABPs are “peptide ligand domains.” The term “peptide liganddomain” means an amino acid sequence which can exist either by itselfand/or within in a larger polypeptide sequence and which binds anotherbiomolecule with specificity. For example, the main blood transportsystem for fatty acids, bilirubin, tryptophan, calcium, steroid hormonesand other physiologically important compounds involves the binding ofthese biomolecules to serum albumin. The binding of these biomoleculesoccurs at discrete sites in the albumin amino acid sequences, i.e., atpeptide ligand domains in serum albumin.

The invention provides compositions for delivering a therapeutic ordiagnostic agent to a disease site in a mammal comprising atherapeutically or diagnostically effective amount of a pharmaceuticalcomposition comprising the therapeutic or diagnostic agent coupled to aSPARC-binding-peptide (“SBP”) and a pharmaceutically acceptable carrier(“inventive compositions” and “inventive methods”). The presentinvention includes compositions and methods wherein the SBP comprises apeptide with the sequence of any one or more of SEQ ID NOs: 1-117, andmost desirably, any one or more of SEQ ID NOS: 1-5, or one or morehomologs of any one of SEQ ID NOs: 1-117.

The term “homolog” means a polypeptide having substantially the sameamino acid sequence as the original sequence and exhibiting relevantproperties that are substantially similar to the properties exhibited bythe original sequence. Illustrative of one such property is the abilityto modulate the tissue distribution of an active agent, wherein ahomolog of SEQ ID NOs: 1-117 would be able to provide a substantiallysimilar level of modulation to that provided by SEQ ID NOs: 1-117. Inthis context, for example and desirably, a homolog of SEQ ID NOs: 1-117exhibiting such substantially similar modulation would provide a bloodlevel of the active agent of at last about 80%, preferably at leastabout 85%, more preferably at least about 90%, and most preferably atleast about 95%, relative to that provided by SEQ ID NOs: 1-117.Alternatively, the term “homolog” also refers to, e.g., a peptidesequence of at least 6 consecutive amino acids, preferably at least 7consecutive amino acids, more preferably at least 8 consecutive aminoacids, even more preferably at least 9 consecutive amino acids, mostpreferably at least 10 consecutive amino acids of any one of SEQ ID NOs:1-112, and most desirably, any one or more of SEQ ID NOs: 1-5.

The compositions and methods provided by the invention also include ABPscomprising SEQ ID NOS: 119 or 120 or both SEQ ID NOS: 119 and 120 andhomologs thereof. Methods in accordance with the invention furtherinclude, e.g., both wherein the SBP and the ABP are in the samepolypeptide and wherein the SBP and the ABP are in differentpolypeptides.

In the context of changes relative to the original sequence, a homologof an original sequence will desirably be at least about 80% identicalto the original sequence, preferably be at least about 90% identical tothe original sequence, even more preferably be at least about 95%identical to the original sequence, and most preferably be at leastabout 99% identical to the original sequence.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window. Additionally, the portion of the polypeptide sequencein the comparison window can comprise additions or deletions (i.e.,gaps) as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percentage of sequenceidentity. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443453.

It is also desirable that where the homologs do not contain identicalamino acids, the mutations result in only conservative amino acidchanges. Accordingly, the residue positions which are not identicaldiffer such that amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity can be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art.

In order to further exemplify what is meant by a “conservative” aminoacid substitution or change in the context of the present invention,Groups A-F are listed below. The replacement of one member of thefollowing groups by another member of the same group is considered to bea “conservative” substitution.

Group A includes leucine, isoleucine, valine, methionine, phenylalanine,serine, cysteinee, threonine, and modified amino acids having thefollowing side chains: ethyl, iso-butyl, —CH2CH2OH, —CH2CH2CH2OH,—CH2CHOHCH3 and CH2SCH3.

Group B includes glycine, alanine, valine, serine, cysteinee, threonine,and a modified amino acid having an ethyl side chain.

Group C includes phenylalanine, phenylglycine, tyrosine, tryptophan,cyclohexylmethyl, and modified amino residues having substituted benzylor phenyl side chains.

Group D includes glutamic acid, aspartic acid, a substituted orunsubstituted aliphatic, aromatic or benzylic ester of glutamic oraspartic acid (e.g., methyl, ethyl, n-propyl, iso-propyl, cyclohexyl,benzyl, or substituted benzyl), glutamine, asparagine, CO—NH-alkylatedglutamine or asparagine (e.g., methyl, ethyl, n-propyl, and iso-propyl),and modified amino acids having the side chain —(CH2)3COOH, an esterthereof (substituted or unsubstituted aliphatic, aromatic, or benzylicester), an amide thereof, and a substituted or unsubstituted N-alkylatedamide thereof.

Group E includes histidine, lysine, arginine, N-nitroarginine,p-cycloarginine, g-hydroxyarginine, N-amidinocitruline, 2-aminoguanidinobutanoic acid, homologs of lysine, homologs of arginine, andornithine.

Group F includes serine, threonine, cysteinee, and modified amino acidshaving C1-C5 straight or branched alkyl side chains substituted with —OHor —SH.

The invention further provides compositions comprising a conjugatemolecule, the conjugate molecule comprising a peptide ligand domainconjugated to an active agent, wherein the peptide ligand domaincomprises up to an additional about 50 amino acids, preferably up to anadditional about 25 amino acids, more preferably up to an additionalabout 15 amino acids, and most preferably up to an additional about 10amino acids added to the amino or carboxyl terminus or both termini. Theresulting polypeptides, which are in accordance with the invention,include polypeptides that are less than 50, less than 40, less than 30,less than 25 or less than 20 amino acids in total length.

The invention further provides compositions comprising a conjugatemolecule, the conjugate molecule comprising a SBP conjugated to anactive agent, wherein there are one or multiple SBP comprising any oneof SEQ ID NOs: 1-137, 141-143, and most desirably, any one or more ofSEQ ID NOS: 1, 2, and 135-137, 141-143.

The invention further provides isolated polynucleotides which encodepolypeptides having the amino acid sequence of peptide ligand bindingdomain including those with said additional amino acid are added to theamino and/or carboxyl termini.

II. Methods of Making Peptides in Accordance with the Invention

The peptide ligand domain-containing polypeptides provided by thepresent invention can be synthesized, detected, quantified and purifiedusing known technologies. For example, cells expressing exogenouspeptide ligand domain-containing polypeptides can be generated byplacing a cDNA under the control of strong promoter/translation startand the vector transfected or transformed into suitable prokaryotic oreukaryotic cells to drive the expression of peptide liganddomain-containing polypeptides by methods well known to those ofordinary skill in the art. Alternatively, peptide liganddomain-containing polypeptides can be made chemically by methods wellknown to those of ordinary skill in the art.

The peptide ligand domain-containing polypeptides can be prepared bystandard solid phase synthesis. As is generally known, peptides of therequisite length can be prepared using commercially available equipmentand reagents following the manufacturers' instructions for blockinginterfering groups, protecting the amino acid to be reacted, coupling,deprotection, and capping of unreacted residues. Suitable equipment canbe obtained, for example, from Applied BioSystems, Foster City, Calif.,or Biosearch Corporation in San Raphael, Calif.

For example, the peptides are synthesized using standard automatedsolid-phase synthesis protocols employing t-butoxycarbonyl-alpha-aminoacids with appropriate side-chain protection. Completed peptide isremoved from the solid phase support with simultaneous side-chaindeprotection using the standard hydrogen fluoride method. Crude peptidesare further purified by semi-preparative reverse phase-HPLC (Vydac C18)using acetonitrile gradients in 0.1% trifluoroacetic acid (TFA). Thepeptides are vacuum dried to remove acetonitrile and lyophilized from asolution of 0.1% TFA in water. Purity is verified by analytical RP-HPLC.The peptides can be lyophilized and then solubilized in either water or0.01M acetic acid at concentrations of 1-2 mg/mL by weight.

The use of the aforementioned synthetic methods is needed if nonencodedamino acids or the D-forms of amino acids occur in the peptides.However, for peptides which are gene-encoded, recourse can also be hadthrough recombinant techniques using readily synthesized DNA sequencesin commercially available expression systems.

The invention accordingly provides for a recombinant vector comprisingthe comprising a elements controlling the expression of a polynucleotidesequence encoding a peptide ligand domain-containing polypeptide. Inaddition, the invention provides for a cell comprising a nucleic acidencoding a peptide ligand domain-containing polypeptide, wherein thecell is a prokaryotic cell or a eukaryotic cell. Methods of microbialand tissue culture are well known to the skilled artisan (see, e.g.,Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York (2001), pp. 16.1-16.54). The inventionthus provides for method of making peptide ligand domain-containingpolypeptides comprising: (a) transforming cells with a nucleic acidencoding the polypeptide of claim 1; (b) inducing the expression of thepolypeptide by the transformed cells; and (c) purifying the polypeptide.

Protein expression is dependent on the level of RNA transcription, whichis in turn regulated by DNA signals. Similarly, translation of mRNArequires, at the very least, an AUG initiation codon, which is usuallylocated within 10 to 100 nucleotides of the 5′ end of the message.Sequences flanking the AUG initiator codon have been shown to influenceits recognition. For example, for recognition by eukaryotic ribosomes,AUG initiator codons embedded in sequences in conformity to a perfect“Kozak consensus” sequence result in optimal translation (see, e.g.,Kozak, J. Molec. Biol. 196: 947-950 (1987)). Also, successful expressionof an exogenous nucleic acid in a cell can require post-translationalmodification of a resultant protein.

The nucleic acid molecules described herein preferably comprise a codingregion operatively linked to a suitable promoter, for example, apromoter functional in eukaryotic cells. Viral promoters, such as,without limitation, the RSV promoter and the adenovirus major latepromoter can be used in the invention. Suitable non-viral promotersinclude, but are not limited to, the phosphoglycerokinase (PGK) promoterand the elongation factor 1α promoter. Non-viral promoters are desirablyhuman promoters. Additional suitable genetic elements, many of which areknown in the art, also can be attached to, or inserted into theinventive nucleic acid and constructs to provide additional functions,level of expression, or pattern of expression.

In addition, the nucleic acid molecules described herein may beoperatively linked to enhancers to facilitate transcription. Enhancersare cis-acting elements of DNA that stimulate the transcription ofadjacent genes. Examples of enhancers which confer a high level oftranscription on linked genes in a number of different cell types frommany species include, without limitation, the enhancers from SV40 andthe RSV-LTR. Such enhancers can be combined with other enhancers whichhave cell type-specific effects, or any enhancer may be used alone.

To optimize protein production in eukaryotic cells, the inventivenucleic acid molecule can further comprise a polyadenylation sitefollowing the coding region of the nucleic acid molecule. Also,preferably all the proper transcription signals (and translationsignals, where appropriate) will be correctly arranged such that theexogenous nucleic acid will be properly expressed in the cells intowhich it is introduced. If desired, the exogenous nucleic acid also canincorporate splice sites (i.e., splice acceptor and splice donor sites)to facilitate mRNA production while maintaining an inframe, full lengthtranscript. Moreover, the inventive nucleic acid molecules can furthercomprise the appropriate sequences for processing, secretion,intracellular localization, and the like.

The nucleic acid molecules can be inserted into any suitable vector.Suitable vectors include, without limitation, viral vectors. Suitableviral vectors include, without limitation, retroviral vectors,alphaviral, vaccinial, adenoviral, adeno associated viral, herpes viral,and fowl pox viral vectors. The vectors preferably have a native orengineered capacity to transform eukaryotic cells, e.g., CHO-K1 cells.Additionally, the vectors useful in the context of the invention can be“naked” nucleic acid vectors (i.e., vectors having little or noproteins, sugars, and/or lipids encapsulating them) such as plasmids orepisomes, or the vectors can be complexed with other molecules. Othermolecules that can be suitably combined with the inventive nucleic acidsinclude without limitation viral coats, cationic lipids, liposomes,polyamines, gold particles, and targeting moieties such as ligands,receptors, or antibodies that target cellular molecules.

The nucleic acid molecules described herein can be transformed into anysuitable cell, typically a eukaryotic cell, such as, e.g., CHO, HEK293,or BHK, desirably resulting in the expression of a peptide liganddomain-containing polypeptide such as, e.g., polypeptide comprising ofSEQ ID NOs: 1-120 or homologs thereof as described herein. The cell canbe cultured to provide for the expression of the nucleic acid moleculeand, therefore, the production of the peptide ligand domain-containingpolypeptide such as, e.g., a polypeptide comprising the amino acidsequence of SEQ ID NOs: 1-120 or homolog thereof as described herein.

Accordingly, the invention provides for a cell transformed ortransfected with an inventive nucleic acid molecule described herein.Means of transforming, or transfecting, cells with exogenous DNAmolecules are well known in the art. For example, without limitation, aDNA molecule is introduced into a cell using standard transformation ortransfection techniques well known in the art such as calcium-phosphateor DEAE-dextran-mediated transfection, protoblast fusion,electroporation, liposomes and direct microinjection (see, e.g.,Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York (2001), pp. 1.1-1.162, 15.1-15.53,16.1-16.54).

Another example of a transformation method is the protoplast fusionmethod, protoplasts derived from bacteria carrying high numbers ofcopies of a plasmid of interest are mixed directly with culturedmammalian cells. After fusion of the cell membranes (usually withpolyethylene glycol), the contents of the bacteria are delivered intothe cytoplasm of the mammalian cells, and the plasmid DNA is transferredto the nucleus.

Electroporation, the application of brief, high-voltage electric pulsesto a variety of mammalian and plant cells leads to the formation ofnanometer-sized pores in the plasma membrane. DNA is taken directly intothe cell cytoplasm either through these pores or as a consequence of theredistribution of membrane components that accompanies closure of thepores. Electroporation can be extremely efficient and can be used bothfor transient expression of clones genes and for establishment of celllines that carry integrated copies of the gene of interest.

Such techniques can be used for both stable and transient transformationof eukaryotic cells. The isolation of stably transformed cells requiresthe introduction of a selectable marker in conjunction with thetransformation with the gene of interest. Such selectable markersinclude genes which confer resistance to neomycin as well as the HPRTgene in HPRT negative cells. Selection can require prolonged culture inselection media, at least for about 2-7 days, preferable for at leastabout 1-5 weeks (see, e.g., Sambrook & Russell, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001),pp. 16.1-16.54).

A peptide ligand domain-containing polypeptide can be expressed andpurified from a recombinant host cell. Recombinant host cells may beprokaryotic or eukaryotic, including but not limited to bacteria such asE. coli, fungal cells such as yeast, insect cells including but, notlimited to, drosophila and silkworm derived cell lines, and mammaliancells and cell lines. When expressing a peptide ligand domain-containingpolypeptide in a cell, e.g., a human cell, whether, in vitro or in vivo,the codons selected for such the polynucleotide encoding the peptide canbe optimized for a given cell type (i.e., species). Many techniques forcodon optimization are known in the art (see, e.g., Jayaraj et al,Nucleic Acids Res. 33(9):3011-6 (2005); Fuglsang et al., Protein Expr.Purif. 31(2):247-9 (2003); Wu et al., “The Synthetic Gene Designer: aFlexible Web Platform to Explore Sequence Space of Synthetic Genes forHeterologous Expression,” csbw, 2005 IEEE Computational SystemsBioinformatics Conference—Workshops (CSBW'05), pp. 258-259 (2005)).

Issues which must be considered for optimal polypeptide expression inprokaryotes include the expression systems used, selection of hoststrain, mRNA stability, codon bias, inclusion body formation andprevention, fusion protein and site-specific proteolysis, compartmentdirected secretion. (see Sorensen et al., Journal of Biotechnology 115(2005) 113-128, which is hereby incorporated by reference).

Expression is normally induced from a plasmid harboured by a systemcompatible genetic background. The genetic elements of the expressionplasmid include origin of replication (ori), an antibiotic resistancemarker, transcriptional promoters, translation initiation regions (TIRs)as well as transcriptional and translational terminators.

Any suitable expression system can be used, for example, Escherichiacoli facilitates protein expression by its relative simplicity,high-density cultivation, the well-known genetics and the large numberof compatible tools, including a variety of available plasmids,recombinant fusion partners and mutant strains, that are available forpolypeptide expression. The E. coli strain or genetic background forrecombinant expression is highly important. Expression strains should bedeficient in the most harmful natural proteases, maintain the expressionplasmid stably and confer the genetic elements relevant to theexpression system (e.g., DE3).

Plasmid copy number is controlled by the origin of replication thatpreferably replicates in a relaxed fashion (Baneyx, 1999). The ColE1replicon present in modern expression plasmids is derived from thepBR322 (copy number 15-20) or the pUC (copy number 500-700) family ofplasmids, whereas the p15A replicon is derived from pACYC184 (copynumber 10-12). The most common drug resistance markers in recombinantexpression plasmids confer resistance to ampicillin, kanamycin,chloramphenicol or tetracycline.

E. coli expression systems include T7 based pET expression system(commercialized by Novagen), lambda PL promoter/cI repressor (e.g.,Invitrogen pLEX), Trc promoter (e.g., Amersham Biosciences pTrc), Tacpromoter (e.g., Amersham Biosciences pGEX) and hybrid lac/T5 (e.g.,Qiagen pQE) and the BAD promoter (e.g., Invitrogen pBAD).

Translation initiation from the translation initiation region (TIR) ofthe transcribed messenger RNA require a ribosomal binding site (RBS)including the Shine-Dalgarno (SD) sequence and a translation initiationcodon. The Shine-Dalgarno sequence is located 7±2 nucleotides upstreamfrom the initiation codon, which is the canonical AUG in efficientrecombinant expression systems. Optimal translation initiation isobtained from mRNAs with the SD sequence UAAGGAGG.

Codon usage in E. coli is reflected by the level of cognateamino-acylated tRNAs available in the cytoplasm. Major codons occur inhighly expressed genes whereas the minor or rare codons tend to be ingenes expressed at low levels. Codons rare in E. coli are often abundantin heterologous genes from sources such as eukaryotes, archaeabacteriaand other distantly related organisms with different codon frequencypreferencies (Kane, 1995). Expression of genes containing rare codonscan lead to translational errors, as a result of ribosomal stalling atpositions requiring incorporation of amino acids coupled to minor codontRNAs (McNulty et al., 2003). Codon bias problems become highlyprevalent in recombinant expression systems, when transcripts containingrare codons in clusters, such as doublets and triplets accumulate inlarge quantities.

Protein activity demands folding into precise three dimensionalstructures. Stress situations such as heat shock impair folding in vivoand folding intermediates tend to associate into amorphous proteingranules termed inclusion bodies.

Inclusion bodies are a set of structurally complex aggregates oftenperceived to occur as a stress response when recombinant protein isexpressed at high rates. Macromolecular crowding of proteins atconcentrations of 200-300 mg/ml in the cytoplasm of E. coli, suggest ahighly unfavorable protein-folding environment, especially duringrecombinant high-level expression (van den Berg et al., 1999). Whetherinclusion bodies form through a passive event occurring by hydrophobicinteraction between exposed patches on unfolded chains or by specificclustering mechanisms is unknown (Villaverde and Carrio, 2003). Thepurified aggregates can be solubilized using detergents like urea andguadinium hydrochloride. Native protein can be prepared by in vitrorefolding from solubilized inclusion bodies either by dilution, dialysisor on-column refolding methods (Middelberg, 2002; Sørensen et al.,2003a).

Refolding strategies might be improved by inclusion of molecularchaperones (Mogk et al., 2002). Optimization of the refolding procedurefor a given protein however require time consuming efforts and is notalways conducive to high product yields. A possible strategy for theprevention of inclusion body formation is the co-overexpression ofmolecular chaperones.

A wide range of protein fusion partners has been developed in order tosimplify the purification and expression of recombinant proteins(Stevens, 2000). Fusion proteins or chimeric proteins usually include apartner or “tag” linked to the passenger or target protein by arecognition site for a specific protease. Most fusion partners areexploited for specific affinity purification strategies. Fusion partnersare also advantageous in vivo, where they might protect passengers fromintracellular proteolysis (Jacquet et al., 1999; Martinez et al., 1995),enhance solubility (Davis et al., 1999; Kapust and Waugh, 1999; Sørensenet al., 2003b) or be used as specific expression reporters (Waldo etal., 1999). High expression levels can often be transferred from aN-terminal fusion partner, to a poorly expressing passenger, mostprobably as a result of mRNA stabilization (Arechaga et al., 2003).Common affinity tags are the polyhistidine tag (His-tag), which iscompatible with immobilized metal affinity chromatography (IMAC) and theglutathione S-transferase (GST) tag for purification on glutathionebased resins. Several other affinity tags exist and have beenextensively reviewed (Terpe, 2003).

Recombinantly expressed proteins can in principle be directed to threedifferent locations namely the cytoplasm, the periplasm or thecultivation medium. Various advantages and disadvantages are related tothe direction of a recombinant protein to a specific cellularcompartment. Expression in the cytoplasm is normally preferable sinceproduction yields are high. Disulfide bond formation is segregated in E.coli and is actively catalyzed in the periplasm by the Dsb system(Rietsch and Beckwith, 1998). Reduction of cysteines in the cytoplasm isachieved by thioredoxin and glutaredoxin. Thioredoxin is kept reduced bythioredoxin reductase and glutaredoxin by glutathione. The low molecularweight glutathione molecule is reduced by glutathione reductase.Disruption of the trxB and gor genes encoding the two reductases, allowthe formation of disulfide bonds in the E. coli cytoplasm.

Cell-based expression systems have drawbacks in terms of the quality andquantity of the proteins produced and are not always appropriate forhigh-throughput production. Many of these shortcomings can becircumvented by the use of cell-free translation systems.

Cell-free systems for in vitro gene expression and protein synthesishave been described for many different prokaryotic and eukaryoticsystems (see Endo & Sawasaki Current Opinion in Biotechnology 2006,17:373-380. Eukaryotic cell-free systems, such as rabbit reticulocytelysate and wheat germ extract, are prepared from crude extractcontaining all the components required for translation of invitro-transcribed RNA templates. Eukaryotic cell-free systems useisolated RNA synthesized in vivo or in vitro as a template for thetranslation reaction (e.g., Rabbit Reticulocyte Lysate Systems or WheatGerm Extract Systems). Coupled eukaryotic cell-free systems combine aprokaryotic phage RNA polymerase with eukaryotic extracts and utilize anexogenous DNA or PCR-generated templates with a phage promoter for invitro protein synthesis (e.g., TNT® Coupled Reticulocyte Lysate

Proteins translated using the TNT® Coupled Systems can be used in manytypes of functional studies. TNT® Coupled Transcription/Translationreactions have traditionally been used to confirm open reading frames,study protein mutations and make proteins in vitro for protein-DNAbinding studies, protein activity assays, or protein-protein interactionstudies. Recently, proteins expressed using the TNT® Coupled Systemshave also been used in assays to confirm yeast two-hybrid interactions,perform in vitro expression cloning (IVEC) and make protein substratesfor enzyme activity or protein modification assays. For a listing ofrecent citations using the TNT® Coupled Systems in various applications,please visit: www.promega.com/citations/

Transcription and translation are typically coupled in prokaryoticsystems; that is, they contain an endogenous or phage RNA polymerase,which transcribes mRNA from an exogenous DNA template. This RNA is thenused as a template for translation. The DNA template may be either agene cloned into a plasmid vector (cDNA) or a PCR(a)-generated template.A ribosome binding site (RBS) is required for templates translated inprokaryotic systems. During transcription, the 5′-end of the mRNAbecomes available for ribosome binding and translation initiation,allowing transcription and translation to occur simultaneously.Prokaryotic systems are available that use DNA templates containingeither prokaryotic promoters (such as lac or tac; E. coli S30 ExtractSystem for Circular and Linear DNA or a phage RNA polymerase promoter;E. coli T7 S30 Extract System for Circular DNA Solubility of a purifiedpeptide ligand domain-containing polypeptide can be improved by methodsknown in the art. For example, to increase the solubility of anexpressed protein (e.g., in E. coli), one can reduce the rate of proteinsynthesis by lowering the growth temperature, using a weaker promoter,using a lower copy number plasmid, lowering the inducer concentration,changing the growth medium as described in Georgiou & Valax (CurrentOpinion Biotechnol. 7:190-197 (1996)). This decreases the rate ofprotein synthesis and usually more soluble protein is obtained. One canalso add prostethic groups or co-factors which are essential for properfolding or for protein stability, or add buffer to control pHfluctuation in the medium during growth, or add 1% glucose to repressinduction of the lac promoter by lactose, which is present in most richmedia (such as LB, 2×YT). Polyols (e.g., sorbitol) and sucrose may alsobe added to the media because the increase in osmotic pressure caused bythese additions leads to the accumulation of osmoprotectants in thecell, which stabilize the native protein structure. Ethanol, lowmolecular weight thiols and disulfides, and NaCl may be added. Inaddition, chaperones and/or foldases may be co-expressed with thedesired polypeptide. Molecular chaperones promote the properisomerization and cellular targeting by transiently interacting withfolding intermediates. E. coli chaperone systems include but, are notlimited to: GroES-GroEL, DnaK-DnaJ-GrpE, CIpB.

Foldases accelerate rate-limiting steps along the folding pathway. Threetypes of foldases play an important role: peptidyl prolyl cis/transisomerases (PPI's), disulfide oxidoreductase (DsbA) and disulfideisomerase (DsbC), protein disulfide isomerase (PDI) which is aneukaryotic protein that catalyzes both protein cysteine oxidation anddisulfide bond isomerization. Co-expression of one or more of theseproteins with the target protein could lead to higher levels of solubletarget protein.

A peptide ligand domain-containing polypeptide can be produced as afusion protein in order to improve its solubility and production. Thefusion protein comprises a peptide ligand domain-containing polypeptideand a second polypeptide fused together in frame. The second polypeptidemay be a fusion partner known in the art to improve the solubility ofthe polypeptide to which it is fused, for example, NusA,bacterioferritin (BFR), GrpE, thioredoxin (TRX) and glutathione-S—transferase (GST). Novagen Inc. (Madison, Wis.) provides the pET 43.1vector series which permit the formation of a NusA-target fusion. DsbAand DsbC have also shown positive effects on expression levels when usedas a fusion partner, therefore can be used to fuse with a peptide liganddomain for achieving higher solubility.

In an aspect of such fusion proteins, the expressed peptide liganddomain-containing polypeptide includes a linker polypeptide comprises aprotease cleavage site comprising a peptide bond which is hydrolyzableby a protease. As a result, the peptide ligand domain in a polypeptidecan be separated from the remainder of the polypeptide after expressionby proteolysis. The linker can comprise one or more additional aminoacids on either side of the bond to which the catalytic site of theprotease also binds (see, e.g., Schecter & Berger, Biochem. Biophys.Res. Commun. 27, 157-62 (1967)). Alternatively, the cleavage site of thelinker can be separate from the recognition site of the protease and thetwo cleavage site and recognition site can be separated by one or more(e.g., two to four) amino acids. In one aspect, the linker comprises atleast about 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 20, about 30, about40, about 50 or more amino acids. More preferably the linker is fromabout 5 to about 25 amino acids in length, and most preferably, thelinker is from about 8 to about 15 amino acids in length.

Some proteases useful according to the invention are discussed in thefollowing references: Hooper et al., Biochem. J. 321: 265-279 (1997);Werb, Cell 91: 439-442 (1997); Wolfsberg et al., J. Cell Biol. 131:275-278 (1995); Murakami & Etlinger, Biochem. Biophys. Res. Comm. 146:1249-1259 (1987); Berg et al., Biochem. J. 307: 313-326 (1995); Smythand Trapani, Immunology Today 16: 202-206 (1995); Talanian et al., J.Biol. Chem. 272: 9677-9682 (1997); and Thornberry et al., J. Biol. Chem.272: 17907-17911 (1997). Cell surface proteases also can be used withcleavable linkers according to the invention and include, but are notlimited to: Aminopeptidase N; Puromycin sensitive aminopeptidase;Angiotensin converting enzyme; Pyroglutamyl peptidase II; Dipeptidylpeptidase IV; N-arginine dibasic convertase; Endopeptidase 24.15;Endopeptidase 24.16; Amyloid precursor protein secretases alpha, betaand gamma; Angiotensin converting enzyme secretase; TGF alpha secretase;TNF alpha secretase; FAS ligand secretase; TNF receptor-I and -IIsecretases; CD30 secretase; KL1 and KL2 secretases; IL6 receptorsecretase; CD43, CD44 secretase; CD16-I and CD16-II secretases;L-selectin secretase; Folate receptor secretase; MMP 1, 2, 3, 7, 8, 9,10, 11, 12, 13, 14, and 15; Urokinase plasminogen activator; Tissueplasminogen activator; Plasmin; Thrombin; BMP-1 (procollagenC-peptidase); ADAM 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11; and, GranzymesA, B, C, D, E, F, G, and H.

An alternative to relying on cell-associated proteases is to use aself-cleaving linker. For example, the foot and mouth disease virus(FMDV) 2A protease may be used as a linker. This is a short polypeptideof 17 amino acids that cleaves the polyprotein of FMDV at the 2A/2Bjunction. The sequence of the FMDV 2A propeptide is NFDLLKLAGDVESNPGP.Cleavage occurs at the C-terminus of the peptide at the finalglycine-proline amino acid pair and is independent of the presence ofother FMDV sequences and cleaves even in the presence of heterologoussequences.

Affinity chromatography can be used alone or in conjunction withion-exchange, molecular sizing, or HPLC chromatographic techniques inthe purification of peptide ligand domain-containing polypeptides. Suchchromatographic approach can be performed using columns or in batchformats. Such chromatographic purification methods are well known in theart.

Additionally, the invention provides for isolated nucleic acids encodingpeptide ligand domain-containing polypeptides with one or more aminoacid substitutions and insertions or deletions of from about 1 to about5 amino acids, preferably from about 1 to about 3 amino acids, morepreferably 1 amino acid, in the SEQ ID NOs: 1-117 sequences, wherein therelevant properties that are substantially similar to the propertiesexhibited by the original sequence.

Mutagenesis can be undertaken by any of several methods known in theart. Generally, mutagenesis can be accomplished by cloning the nucleicacid sequence into a plasmid or some other vector for ease ofmanipulation of the sequence. Then, a unique restriction site at whichfurther nucleic acids can be added into the nucleic acid sequence isidentified or inserted into the nucleic acid sequence. A double-strandedsynthetic oligonucleotide generally is created from overlappingsynthetic single-stranded sense and antisense oligonucleotides such thatthe double-stranded oligonucleotide incorporates the restriction sitesflanking the target sequence and, for instance, can be used toincorporate replacement DNA. The plasmid or other vector is cleaved withthe restriction enzyme, and the oligonucleotide sequence havingcompatible cohesive ends is ligated into the plasmid or other vector toreplace the original DNA.

Other means of in vitro site-directed mutagenesis are known to thoseskilled in the art, and can be accomplished (in particular, using anoverlap-extension polymerase chain reaction (PCR), see, e.g., Parikh &Guengerich, Biotechniques 24:428-431 (1998)). Complementary primersoverlapping the site of change can be used to PCR amplify the wholeplasmid in a mixture containing 500 mM dNTPs, 2 units of Pfu polymerase,250 ng each of sense and antisense primers, and 200 ng of plasmid DNAcomprising a sequence encoding Peptide ligand domain-containingpolypeptide. The PCR desirably involves 18 cycles with an extension timeof 2.5 minutes for each Kb of DNA. The PCR products can be treated withDpnI (which only digests the adenine-methylated plasmid DNA) andtransformed into Escherichia coli DH5α cells. Transformants can bescreened by restriction enzyme digestion for incorporation of thechanges, which then can be confirmed by DNA sequence analysis.

Suitable methods of protein detection and quantification of peptideligand domain-containing polypeptides include Western blot,enzyme-linked immunosorbent assay (ELISA), silver staining, the BCAassay (see, e.g., Smith et al., Anal. Biochem., 150, 76-85 (1985)), theLowry protein assay (described in, e.g., Lowry et al., J. Biol. Chem.,193, 265-275 (1951)) which is a colorimetric assay based onprotein-copper complexes, and the Bradford protein assay (described in,e.g., Bradford et al., Anal. Biochem., 72, 248 (1976)) which dependsupon the change in absorbance in Coomassie Blue G-250 upon proteinbinding. Once expressed, the peptide ligand domain-containingpolypeptides can be purified by traditional purification methods such asionic exchange, size exclusion, or C18 chromatography.

III. Methods of Coupling Peptide Ligand Domains

Methods for “coupling” (or “conjugation” or “cross-linking”) of suitableactive agents such as, e.g., therapeutics, chemotherapeutics,radionuclides, polypeptides, and the like, to peptide liganddomain-containing polypeptide are well described in the art. Inpreparing the conjugates provided herein, the active agent is linkedeither directly or indirectly peptide ligand domain by any methodpresently known in the art for attaching two moieties, so long as theattachment of the conjugating or coupling moiety to the peptide liganddomain does not substantially impede its function of the peptide liganddomain or substantially impede the function of the active agent. Thecoupling can be by any suitable means, including, but are not limitedto, ionic and covalent bonds, and any other sufficiently stableassociation, whereby the targeted agent's distribution will bemodulated.

Numerous heterobifunctional cross-linking reagents that are used to formcovalent bonds between amino groups and thiol groups and to introducethiol groups into proteins, are known to those of skill in this art(see, e.g., Cumber et al. (1992) Bioconjugate Chem. 3′:397 401; Thorpeet al. (1987) Cancer Res. 47:5924 5931; Gordon et al. (1987) Proc. Natl.Acad. Sci. 84:308 312; Walden et al. (1986) J. Mol. Cell. Immunol. 2:191197; Carlsson et al. (1978) Biochem. J. 173: 723 737; Mahan et al.(1987) Anal. Biochem. 162:163 170; Wawryznaczak et al. (1992) Br. J.Cancer 66:361 366; Fattom et al. (1992) Infection & Immun. 60:584 589).These reagents may be used to form covalent bonds between a peptideligand domain or a peptide ligand domain-containing polypeptide and anyof the active agents disclosed herein. These reagents include, but arenot limited to: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP;disulfide linker); sulfosuccinimidyl6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP);succinimidyloxycarbonyl-α-methyl benzyl thiosulfate (SMBT, hindereddisulfate linker); succinimidyl6-[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP);sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC); succinimidyl 3-(2-pyridyldithio)butyrate (SPDB; hindereddisulfide bond linker); sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3-dithiopropionate(SAED); sulfo-succinimidyl 7-azido-4-methylcoumarin-3-acetate (SAMCA);sulfosuccinimidyl6-[alpha-methyl-alpha-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-SMPT); 1,4-di-[3′-(2′-pyridyldithio)propionamido]butane(DPDPB); 4-succinimidyloxycarbonyl-α-methyl-α.-(2-pyridylthio)-toluene(SMPT, hindered disulfate linker);sulfosuccinimidyl6[α.-methyl-α.-(2-pyridyldithio)toluamido]hexa-noate(sulfo-LC-SMPT); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB; thioether linker);sulfosuccinimidyl(4-iodoacetyl)amino benzoate (sulfo-SIAB);succinimidyl-4(p-maleimidophenyl)butyrate (SMPB);sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-SMPB);azidobenzoyl hydrazide (ABH).

Other heterobifunctional cleavable coupling agents include,N-succinimidyl(4-iodoacetyl)-aminobenzoate; sulfosuccinimydil(4-iodoacetyl)-aminobenzoate;4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)-toluene;sulfosuccinimidyl-6-[a-methyl-a-(pyridyldithiol)-toluamido]hexanoate;N-succinimidyl-3-(-2-pyridyldithio)-proprionate; succinimidyl6[3(-(-2-pyridyldithio)-proprionamido]hexanoate; sulfosuccinimidyl6[3(-(-2-pyridyldithio)-propionamido]hexanoate;3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine. Further exemplarybifunctional linking compounds are disclosed in U.S. Pat. Nos.5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877.

Alternatively, e.g., polypeptide suflhydryl groups can be used forconjugation. In addition, sugar moieties bound to glycoproteins, e.g.,antibodies can be oxidized to form aldehydes groups useful in a numberof coupling procedures known in the art. The conjugates formed inaccordance with the invention can be stable in vivo or labile, such asenzymatically degradeable tetrapeptide linakages or acid-labilecis-aconityl or hydrazone linkages.

The peptide ligand domain-containg polypeptide is optionally linked tothe active agent via one or more linkers. The linker moiety is selecteddepending upon the properties desired. For example, the length of thelinker moiety can be chosen to optimize the kinetics and specificity ofligand binding, including any conformational changes induced by bindingof the ligand to a target receptor. The linker moiety should be longenough and flexible enough to allow the polypeptide ligand moiety andthe target cell receptor to freely interact. If the linker is too shortor too stiff, there may be steric hindrance between the polypeptideligand moiety and the cell toxin. If the linker moiety is too long, theactive agent may be degraded in the process of production, or may notdeliver its desired effect to the target cell effectively.

Any suitable linker known to those of skill in the art can be usedherein. Generally a different set of linkers will be used in conjugatesthat are fusion proteins from linkers in chemically-produced conjugates.Linkers and linkages that are suitable for chemically linked conjugatesinclude, but are not limited to, disulfide bonds, thioether bonds,hindered disulfide bonds, and covalent bonds between free reactivegroups, such as amine and thiol groups. These bonds are produced usingheterobifunctional reagents to produce reactive thiol groups on one orboth of the polypeptides and then reacting the thiol groups on onepolypeptide with reactive thiol groups or amine groups to which reactivemaleimido groups or thiol groups can be attached on the other. Otherlinkers include, acid cleavable linkers, such as bismaleimideothoxypropane, acid labile-transferrin conjugates and adipic aciddiihydrazide, that would be cleaved in more acidic intracellularcompartments; cross linkers that are cleaved upon exposure to UV orvisible light and linkers. In some embodiments, several linkers may beincluded in order to take advantage of desired properties of eachlinker. Chemical linkers and peptide linkers may be inserted bycovalently coupling the linker to the peptide ligand domain-containgpolypeptide and the targeted agent. The heterobifunctional agents,described below, may be used to effect such covalent coupling. Peptidelinkers may also be linked by expressing DNA encoding the linker andpeptide ligand domain, linker and active agent, or peptide liganddomain, linker and active agent as a fusion protein. Flexible linkersand linkers that increase solubility of the conjugates are contemplatedfor use, either alone or with other linkers are also contemplatedherein.

Accordingly, linkers can include, but are not limited to, peptidiclinkages, amino acid and peptide linkages, typically containing betweenone and about 30 amino acids, more preferably between about 10 and 30amino acids. Alternatively, chemical linkers, such as heterobifunctionalcleavable cross-linkers, including but are not limited to,N-succinimidyl(4-iodoacetyl)-aminobenzoate,sulfosuccinimydil(4-iodoacetyl)-aminobenzoate,4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)toluene,sulfosuccinimidyl-6-a-methyl-a-(pyridyldithiol)-toluamido)hexanoate,N-succinimidyl-3-(-2-pyridyldithio)-proprionate, succinimidyl6(3(-(-2-pyridyldithio)-proprionamido)hexanoate, sulfosuccinimidyl6(3(-(-2-pyridyldithio)-propionamido)hexanoate,3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine.

Other linkers, include trityl linkers, particularly, derivatized tritylgroups to generate a genus of conjugates that provide for release oftherapeutic agents at various degrees of acidity or alkalinity. Theflexibility thus afforded by the ability to preselect the pH range atwhich the therapeutic agent will be released allows selection of alinker based on the known physiological differences between tissues inneed of delivery of a therapeutic agent (see, e.g., U.S. Pat. No.5,612,474). For example, the acidity of tumor tissues appears to belower than that of normal tissues.

Acid cleavable linkers, photocleavable and heat sensitive linkers mayalso be used, particularly where it may be necessary to cleave thetargeted agent to permit it to be more readily accessible to reaction.Acid cleavable linkers include, but are not limited to,bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see,e.g., Fattom et al. (1992) Infection & Immun. 60:584 589) and acidlabile transferrin conjugates that contain a sufficient portion oftransferrin to permit entry into the intracellular transferrin cyclingpathway (see, e.g., Welhoner et al. (1991) J. Biol. Chem. 266:43094314).

Photocleavable linkers are linkers that are cleaved upon exposure tolight (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104 107,which linkers are herein incorporated by reference), thereby releasingthe targeted agent upon exposure to light. Photocleavable linkers thatare cleaved upon exposure to light are known (see, e.g., Hazum et al.(1981) in Pept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp.105 110, which describes the use of a nitrobenzyl group as aphotocleavable protective group for cysteine; Yen et al. (1989)Makromol. Chem. 190:69 82, which describes water soluble photocleavablecopolymers, including hydroxypropylmethacrylamide copolymer, glycinecopolymer, fluorescein copolymer and methylrhodamine copolymer;Goldmacher et al. (1992) Bioconj. Chem. 3:104 107, which describes across-linker and reagent that undergoes photolytic degradation uponexposure to near UV light (350 nm); and Senter et al. (1985) Photochem.Photobiol 42:231 237, which describes nitrobenzyloxycarbonyl chloridecross linking reagents that produce photocleavable linkages), therebyreleasing the targeted agent upon exposure to light. Such linkers wouldhave particular use in treating dermatological or ophthalmic conditionsthat can be exposed to light using fiber optics. After administration ofthe conjugate, the eye or skin or other body part can be exposed tolight, resulting in release of the targeted moiety from the conjugate.Such photocleavable linkers are useful in connection with diagnosticprotocols in which it is desirable to remove the targeting agent topermit rapid clearance from the body of the animal.

IV. The Invention Provides a Plurality of Active Agents

The various aspects of the present invention contemplate that thepeptide ligand domain-containing polypeptide is coupled to an activeagent, i.e., a therapeutic or diagnostic agent.

As used herein, the term “therapeutic agent” refers to a chemicalcompound, a biological macromolecule, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularlymammalian) cells or tissues that are suspected of having therapeuticproperties, e.g., chemotherapeutic agent or radiotherapy agent. The term“therapeutic” as used herein refers to ameliorating the effects ofcuring or preventing (illustrated by the prevention or lessening thechance of a targeted disease, e.g., cancer or other proliferativedisease) a disease or related condition afflicting a subject mammal.Curative therapy refers alleviating, in whole or in part, an existingdisease or condition in a mammal.

The agent can be purified, substantially purified or partially purified.Further, such a therapeutic agent can be in or associated with aliposome or immunoliposome and the conjugation can be directly to theagent or to the liposome/immunoliposome. A “liposome” is a small vesiclecomposed of various types of lipids, phospholipids and/or surfactantwhich is useful for delivery of a drug (e.g., drugs, antibodies,toxins). The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

Illustrative of the therapeutic agents which can be coupled to thepeptide ligand domain-containing polypeptide in the manner contemplatedby the present invention include, without limitation, chemotherapeuticagents (e.g., docetaxel, paclitaxel, taxanes and platinum compounds),antifolates, antimetabolites, antimitotics, DNA damaging agents,proapoptotics, differentiation inducing agents, antiangiogenic agents,antibiotics, hormones, peptides, antibodies, tyrosine kinase inhibitors,biologically active agents, biological molecules, radionuclides,adriamycin, ansamycin antibiotics, asparaginase, bleomycin, busulphan,cisplatin, carboplatin, carmustine, capecitabine, chlorambucil,cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin,daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide,epothilones, floxuridine, fludarabine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, mercaptopurine, meplhalan, methotrexate, rapamycin(sirolimus), mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel,pamidronate, pentostatin, plicamycin, procarbazine, rituximab,streptozocin, teniposide, thioguanine, thiotepa, taxanes, vinblastine,vincristine, vinorelbine, taxol, combretastatins, discodermolides,transplatinum, tyrosine kinase inhibitors (genistein), and otherchemotherapeutic agents.

As used herein, the term “chemotherapeutic agent” refers to an agentwith activity against cancer, neoplastic, and/or proliferative diseases.Preferred chemotherapeutic agents include docetaxel and paclitaxel asparticles comprising albumin wherein more than 50% of thechemotherapeutic agent is in nanoparticle form. Most preferably, thechemotherapeutic agent comprises particles of albumin-bound paclitaxel,e.g., Abraxane®.

Suitable therapeutic agents also include, e.g., biologically activeagents (TNF, of tTF), radionuclides (131I, 90Y, 111In, 211At, 32P andother known therapeutic radionuclides), antiangiogenesis agents(angiogenesis inhibitors, e.g., INF-alpha, fumagillin, angiostatin,endostatin, thalidomide, and the like), other biologically activepolypeptides, therapy sensitizers, antibodies, lectins, and toxins.

Suitable diseases for the application of the invention include malignantand premalignant conditions, as well as proliferative disease, includingbut, not limited to, where the proliferative diseases is, e.g., benignprostatic hyperplasia, endometriosis, endometrial hyperplasia,atherosclerosis, psoriasis, an immunologic proliferation or aproliferative renal glomerulopathy.

The term “therapeutically effective amount” it is meant an amount thatreturns to normal, either partially or completely, physiological orbiochemical parameters associated with or causative of a disease orcondition. A clinician skilled in the art should be able to determineamount of the pharmaceutical composition that will be therapeuticallyeffective relative to a particular disease or condition. By way ofexample, and in accordance with a preferred embodiment wherein thetherapeutic agent is paclitaxel, the paclitaxel dose administered canrange from about 30 mg/m² to about 1000 mg/m² with a dosing cycle ofabout 3 weeks (i.e., administration of the paclitaxel dose once everyabout three weeks), desirably from about 50 mg/m² to about 800 mg/m²,preferably from about 80 mg/m² to about 700 mg/m², and most preferablyfrom about 250 mg/m² to about 300 mg/m² with a dosing cycle of about 3weeks, preferably a cycle of about 2 weeks, more preferably weeklycycles.

The present invention also has diagnostic aspects. For example, thediagnostic agent can be a tracer or label, including, withoutlimitation, radioactive agents, MRI contrast agents, X-ray contrastagents, ultrasound contrast agents, and PET contrast agents. Thecoupling of these agents, described in connection with therapeuticagents, is also contemplated by this aspect of the invention. Further,the term “diagnostically effective amount” is an amount of thepharmaceutical composition that in relevant clinical settings allows fora reasonably accurate determination of the presence and/or extent ofabnormal proliferative, hyperplastic, remodeling, inflammatory activityin tissues and organs. For example, the condition “diagnosed” inaccordance with the invention can be a benign or malignant tumor.

The diagnostic agents taught herein include polypeptides, such asantibodies, which can be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

The delivery of therapeutic or diagnostic agents to a tumor or otherdisease site by inventive compositions and methods can be monitored andmeasured by any suitable method including, e.g., adding a radioactivelabel or radio-opaque label to the composition and imaging as isappropriate and well known to those of ordinary skill in the art. Thesequesteration of compositions in the plasma compartment can bemonitored by any suitable method including, e.g., venupuncture.

Further, and in a related aspect, the invention provides a method ofpredicting or determining a tumor's response to a chemotherapeuticagent, as well as a method of predicting or determining a proliferativedisease's response to a chemotherapeutic agent or treating aproliferative disease, including but, not limited to, where theproliferative diseases is, e.g., benign prostatic hyperplasia,endometriosis, endometrial hyperplasia, atherosclerosis, psoriasis,immunologic proliferation or a proliferative renal glomerulopathy.

V. The Invention Provides Fusion Proteins which Couple Peptide LigandDomains to Polypeptide Active Agents

The present invention further contemplates the coupling of peptideligand domains to polypeptide active agents in fusion proteins. Forexample, and without limitation, peptide ligand domain sequences can befused upstream or downstream of diagnostically useful protein domains(such as hapten, GFP), a therapy sensitizer, active protein domains(e.g., without limitation, tTF, TNF, Smar1 derived p44 peptide,interferon, TRAIL, Smac, VHL, procaspase, caspase, and IL-2) or toxin(e.g., without limitation, ricin, PAP, Diphtheria toxin, Pseudomonasexotoxin)

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving at least two portions covalently linked together, where each ofthe portions is a polypeptide having a different property. The propertycan be a biological property, such as activity in vitro or in vivo. Theproperty can also be a simple chemical or physical property, such asbinding to a target molecule, catalysis of a reaction, and the like. Theportions can be linked directly by a single peptide bond or through apeptide linker containing one or more amino acid residues. Generally,the portions and the linker will be in reading frame with each other.

VI. Antibody or Antibody Fragment Active Agents

In a particular aspect of the invention, the therapeutic agent can be anantibody or antibody fragment which mediates one or more of complementactivation, cell mediated cytotoxicity, apoptosis, necrotic cell death,and opsinization.

The term “antibody” herein is includes, without limitation, monoclonalantibodies, polyclonal antibodies, dimers, multimers, multispecificantibodies (e.g., bispecific antibodies). Antibodies can be murine,human, humanized, chimeric, or derived from other species. An antibodyis a protein generated by the immune system that is capable ofrecognizing and binding to a specific antigen. A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen canhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) orsubclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) of immunoglobulinmolecule. The immunoglobulins can be derived from any species.

“Antibody fragments” comprise a portion of a full length antibody, whichmaintain the desired biological activity. “Antibody fragments” are oftenthe antigen binding or variable region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies;linear antibodies; fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, CDR (complementary determiningregion), and epitope-binding fragments of any of the above whichimmunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. However, othernon-antigen-binding portions of antibodies can be “antibody fragments”as meant herein, e.g., without limitation, an antibody fragment can be acomplete or partial Fc domain.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen-binding sequences derived from a non-humanprimate (e.g., Old World Monkey or Ape) and human constant regionsequences.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express Fc.gamma.RIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. To assess ADCC activity of a molecule of interest,an in vitro ADCC assay can be performed (U.S. Pat. No. 55,003,621; U.S.Pat. No. 5,821,337). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest can be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA), 95:652-656 (1998).

An antibody which “induces cell death” is one which causes a viable cellto become nonviable. Cell death in vitro can be determined in theabsence of complement and immune effector cells to distinguish celldeath induced by antibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement dependent cytotoxicity (CDC). Thus, the assay for cell deathcan be performed using heat inactivated serum (i.e., in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the antibody is able to induce cell death, loss of membraneintegrity as evaluated by uptake of propidium iodide (PI), trypan blueor 7AAD can be assessed relative to untreated cells. Cell death-inducingantibodies are those which induce PI uptake in the PI uptake assay inBT474 cells.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies).

VII. Method of Modulating the Distribution of Active Agents

Another aspect of the present invention takes advantage of theproperties of the peptide ligand domain-containing conjugates disclosedherein to provide methods for modulating the distribution of an activeagent within the tissue of an animal comprising administering to theanimal a composition comprising a conjugate molecule which comprises apeptide ligand domain conjugated to an active agent, wherein the peptideligand domain comprises a peptide of the SEQ ID NOs: 1-137, 139 or 140,or 141-143 or homologs thereof, and wherein the administration of thecomposition to an animal results in a tissue distribution of the activeagent which is different from the tissue distribution obtained uponadministration of the active agent alone.

The compositions and methods of the present invention desirably providefor modulated tissue distribution of the active agent to a disease site.This desirably manifests itself in providing a concentration of theactive agent at a disease site, and/or an increased or prolonged(half-life) blood level of the active agent, which is greater than thatwhich would be provided if the active agent (in unconjugated form) wasadministered to the animal. This modulation may also manifest itself byenhancing the rate of tissue uptake of the conjugated peptide molecule,increasing the retention of the molecule at its target site, ie. at thetumor, enhancing the rate of diffusion of the conjugated peptidemolecule in the tissue, and/or enhancing the distribution of theconjugated peptide molecule through the tissue, and matching the rate oftissue uptake of the conjugated peptide molecule to the rate ofinternalization of one or more tissue receptors. Such enhancements canbe measured by any suitable method known in the art including, withoutlimitation, the detection, localization and relative quantization ofsuitably labeled active agent, e.g., using radiographic, microscopic,chemical, immunologic or MRI techniques.

By “enhancing the rate” it is meant a rate that is that is at leastabout 33% greater, preferably at least about 25% greater, morepreferably at least about 15% greater, most preferably at least about10% greater. By a “greater concentration at a disease site” it is meanta concentration of the active agent in the conjugate at a disease sitethat is at least about 33% greater, preferably at least about 25%greater, more preferably at least about 15% greater, most preferably atleast about 10% greater than the concentration of the unconjugatedactive agent at a comparable disease site.

Suitable disease sites include, without limitation, the sites ofabnormal conditions of proliferation, tissue remodeling, hyperplasia,exaggerated wound healing in any bodily tissue including soft tissue,connective tissue, bone, solid organs, blood vessel and the like. Morespecific examples of such diseases include cancer, diabetic or otherretinopathy, inflammation, fibrosis, arthritis, restenosis in bloodvessels or artificial blood vessel grafts or intravascular devices andthe like, cataract and macular degeneration, osteoporosis and otherdiseases of the bone, atherosclerosis and other diseases wherecalcification is frequently observed.

In a preferred aspect, the invention provides methods of diagnosingand/or treating a tumor, wherein the tumor is selected from the groupconsisting of oral cavity tumors, pharyngeal tumors, digestive systemtumors, the respiratory system tumors, bone tumors, cartilaginoustumors, bone metastases, sarcomas, skin tumors, melanoma, breast tumors,the genital system tumors, urinary tract tumors, orbital tumors, brainand central nervous system tumors, gliomas, endocrine system tumors,thyroid tumors, esophageal tumors, gastric tumors, small intestinaltumors, colonic tumors, rectal tumors, anal tumors, liver tumors, gallbladder tumors, pancreatic tumors, laryngeal tumors, tumors of the lung,bronchial tumors, non-small cell lung carcinoma, small cell lungcarcinoma, uterine cervical tumors, uterine corpus tumors, ovariantumors, vulvar tumors, vaginal tumors, prostate tumors, prostaticcarcinoma, testicular tumors, tumors of the penis, urinary bladdertumors, tumors of the kidney, tumors of the renal pelvis, tumors of theureter, head and neck tumors, parathyroid cancer, Hodgkin's disease,Non-Hodgkin's lymphoma, multiple myeloma, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronicmyeloid leukemia. In addition, the invention provides for method ofpredicting or determining a tumor's response to a chemotherapeuticagent, methods of treating a tumor, and kits for predicting the responseof a mammalian tumor to a chemotherapeutic agent, wherein the tumor is asarcoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma,small cell carcinoma, basal cell carcinoma, clear cell carcinoma,oncytoma or combinations thereof.

In another aspect, the invention provides compositions and methods ofuse of said compositions, wherein administering the composition to ananimal results in a blood level of the active agent which is greaterthan the blood level obtained upon administration of the active agentalone. Any suitable measure of the active agent's blood level can beused, including without limitation, C_(max), C_(min), and AUC. By“greater than the blood level obtained upon administration of the activeagent alone” it is meant a blood level that is at least about 33%greater, preferably at least about 25% greater, more preferably at leastabout 15% greater, most preferably at least about 10% greater.

In yet another aspect, the invention provides compositions and methodsof use of said compositions, wherein the administration of thecomposition to an animal results in a blood level half-life of theactive agent which is greater than the blood level half-life obtainedupon administration of the active agent alone. By “greater than theblood half-life obtained upon administration of the active agent alone”it is meant a half-life that is at least about 33% greater, preferablyat least about 25% greater, more preferably at least about 15% greater,most preferably at least about 10% greater.

VIII. Formulations and Administration

For use in vivo, the active agent coupled a peptide ligand domain, suchas the SEQ ID NOs: 1-117 and homologs thereof, is desirably isformulated into a pharmaceutical composition comprising aphysiologically acceptable carrier. Any suitable physiologicallyacceptable carrier can be used within the context of the invention,depending on the route of administration. Those skilled in the art willappreciate those carriers that can be used in to provide apharmaceutical composition suitable for the desired method ofadministration.

The administration of the pharmaceutical compositions of the presentinvention can be accomplished via any suitable route including, but notlimited to, intravenous, subcutaneous, intramuscular, intraperitoneal,intratumoral, oral, rectal, vaginal, intravesical, and inhalationaladministration, with intravenous and intratumoral administration beingmost preferred. The composition can further comprise any other suitablecomponents, especially for enhancing the stability of the compositionand/or its end use. Accordingly, there is a wide variety of suitableformulations of the composition of the invention. The followingformulations and methods are merely exemplary and are in no waylimiting.

The pharmaceutical compositions can also include, if desired, additionaltherapeutic or biologically-active agents. For example, therapeuticfactors useful in the treatment of a particular indication can bepresent. Factors that control inflammation, such as ibuprofen orsteroids, can be part of the composition to reduce swelling andinflammation associated with in vivo administration of thepharmaceutical composition and physiological distress.

The carrier typically will be liquid, but also can be solid, or acombination of liquid and solid components. The carrier desirably isphysiologically acceptable (e.g., a pharmaceutically orpharmacologically acceptable) carrier (e.g., excipient or diluent).Physiologically acceptable carriers are well known and are readilyavailable. The choice of carrier will be determined, at least in part,by the location of the target tissue and/or cells, and the particularmethod used to administer the composition.

Typically, such compositions can be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for using toprepare solutions or suspensions upon the addition of a liquid prior toinjection can also be prepared; and the preparations can also beemulsified. The pharmaceutical formulations suitable for injectable useinclude sterile aqueous solutions or dispersions; formulationscontaining known protein stabilizers and lyoprotectants, formulationsincluding sesame oil, peanut oil or aqueous propylene glycol, andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases the formulation must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxycellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The peptide ligand domain-containing conjugate, such as can beformulated into a composition in a neutral or salt form.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such as organic acids as acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups also can bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, procaine and the like.

Formulations suitable for parenteral administration include aqueous andnon aqueous, isotonic sterile injection solutions, which can containanti oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit dose or multi dose sealedcontainers, such as ampules and vials, and can be stored in a freezedried (lyophilized) condition requiring only the addition of a sterileliquid excipient, for example, water, for injections, immediately priorto use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described. In a preferred embodiment of the invention, thepeptide ligand domain-containing conjugate is formulated for injection(e.g., parenteral administration). In this regard, the formulationdesirably is suitable for intratumoral administration, but also can beformulated for intravenous injection, intraperitoneal injection,subcutaneous injection, and the like.

The invention also provides, if desirable, embodiments in which thepeptide ligand domain-containing conjugate (i.e., the peptide liganddomain-containing polypeptide conjugated to an active agent) is furtherconjugated to polyethylene glycol (PEG). PEG conjugation can increasethe circulating half-life of these polypeptides, reduce thepolypeptide's immunogenicity and antigenicity, and improve theirbioactivity. If used, any suitable method of PEG conjugation can beused, including but not limited to, reacting methoxy-PEG with apeptide's available amino group(s) or other reactive sites such as,e.g., histidines or cysteines. In addition, recombinant DNA approachescan be used to add amino acids with PEG-reactive groups to the peptideligand domain-containing conjugate. Further, releasable and hybridPEG-ylation strategies can be used in accordance with the aspects of thepresent invention, such as the PEG-ylation of polypeptide, wherein thePEG molecules added to certain sites in the peptide liganddomain-containing conjugatemolecule are released in vivo. Examples ofPEG conjugation methods are known in the art. See, e.g., Greenwald etal., Adv. Drug Delivery Rev. 55:217-250 (2003).

Formulations suitable for administration via inhalation include aerosolformulations. The aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also can be formulated as non pressurizedpreparations, for delivery from a nebulizer or an atomizer.

Formulations suitable for anal administration can be prepared assuppositories by mixing the active ingredient with a variety of basessuch as emulsifying bases or water soluble bases. Formulations suitablefor vaginal administration can be presented as pessaries, tampons,creams, gels, pastes, foams, or spray formulas containing, in additionto the active ingredient, such carriers as are known in the art to beappropriate.

In addition, the composition of the invention can comprise additionaltherapeutic or biologically active agents. For example, therapeuticfactors useful in the treatment of a particular indication can bepresent. Factors that control inflammation, such as ibuprofen orsteroids, can be part of the composition to reduce swelling andinflammation associated with in vivo administration of thepharmaceutical composition and physiological distress.

In the case of inhalational therapy, the pharmaceutical composition ofthe present invention is desirably in the form of an aerosol. Aerosoland spray generators for administering the agent if in solid form areavailable. These generators provide particles that are respirable orinhalable, and generate a volume of aerosol containing a predeterminedmetered dose of a medicament at a rate suitable for humanadministration. Examples of such aerosol and spray generators includemetered dose inhalers and insufflators known in the art. If in liquidform, the pharmaceutical compositions of the invention can beaerosolized by any suitable device.

When used in connection with intravenous, intraperitoneal orintratumoral administration, the pharmaceutical composition of theinvention can comprise sterile aqueous and non-aqueous injectionsolutions, suspensions or emulsions of the active compound, whichpreparations are preferably isotonic with the blood of the intendedrecipient. These preparations can contain one or more of anti-oxidants,buffers, surfactants, cosolvents, bacteriostats, solutes which renderthe compositions isotonic with the blood of the intended recipient, andother formulation components known in the art. Aqueous and non-aqueoussterile suspensions can include suspending agents and thickening agents.The compositions can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials.

The methods of the present invention can also be part of combinationtherapy. The phrase “combination therapy” refers to administering atherapeutic agent in accordance with the invention together with anothertherapeutic composition in a sequential or concurrent manner such thatthe beneficial effects of this combination are realized in the mammalundergoing therapy.

XI. The Invention is Applicable to Many Conditions

The compositions and methods of the invention are suitable for use indiagnosing or treating various diseases including, but not limited to,wherein the disease site is, abnormal conditions of proliferation,tissue remodeling, hyperplasia, exaggerated wound healing in any bodilytissue including soft tissue, connective tissue, bone, solid organs,blood vessel and the like. More specific examples of such diseasesinclude cancer, diabetic or other retinopathy, inflammation, fibrosis,arthritis, restenosis in blood vessels or artificial blood vessel graftsor intravascular devices and the like, cataract and maculardegeneration, osteoporosis and other diseases of the bone,atherosclerosis and other diseases where calcification is frequentlyobserved.

In a preferred aspect, the invention provides methods of diagnosingand/or treating a tumor, wherein the tumor is selected from the groupconsisting of oral cavity tumors, pharyngeal tumors, digestive systemtumors, the respiratory system tumors, bone tumors, cartilaginoustumors, bone metastases, sarcomas, skin tumors, melanoma, breast tumors,the genital system tumors, urinary tract tumors, orbital tumors, brainand central nervous system tumors, gliomas, endocrine system tumors,thyroid tumors, esophageal tumors, gastric tumors, small intestinaltumors, colonic tumors, rectal tumors, anal tumors, liver tumors, gallbladder tumors, pancreatic tumors, laryngeal tumors, tumors of the lung,bronchial tumors, non-small cell lung carcinoma, small cell lungcarcinoma, uterine cervical tumors, uterine corpus tumors, ovariantumors, vulvar tumors, vaginal tumors, prostate tumors, prostaticcarcinoma, testicular tumors, tumors of the penis, urinary bladdertumors, tumors of the kidney, tumors of the renal pelvis, tumors of theureter, head and neck tumors, parathyroid cancer, Hodgkin's disease,Non-Hodgkin's lymphoma, multiple myeloma, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronicmyeloid leukemia. In addition, the invention provides for method ofpredicting or determining a tumor's response to a chemotherapeuticagent, methods of treating a tumor, and kits for predicting the responseof a mammalian tumor to a chemotherapeutic agent, wherein the tumor is asarcoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma,small cell carcinoma, basal cell carcinoma, clear cell carcinoma,oncytoma or combinations thereof.

The invention provides for embodiments wherein the disease is in amammal, including but not limited to, a human.

X. Kits

The invention provides kits for the treatment of tumors comprising apharmaceutical formulation and instructions for use of the formulationin the treatment of tumors, wherein the pharmaceutical formulationcomprises a conjugate molecule which comprises a peptide ligand domainconjugated to an active agent, and wherein the peptide ligand domaincomprises a peptide of the SEQ ID NOs: 1-137, 139 or 140, or 141-143, ora homolog thereof, wherein the peptide ligand domain has an affinity forhuman serum albumin characterized by an equilibrium dissociationconstant (Kd) of about 700 μM or less, and, optionally, wherein theconjugate molecule further comprises a second peptide ligand domain, andinstructions for use of said kits (e.g., FDA approved package inserts).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This Example demonstrates the identification of SPARC binding peptidesusing the phage display technology and the incorporation of such SPARCbinding peptides into molecules for tumor therapy.

Specifically, a major goal was to generate a molecule with a SPARCbinding peptide conjugated to a therapeutic or diagnostic agent. Inparticular, the goal was to generate a SPARC binding peptide-Fc fusionprotein (FIG. 1), where the antibody Fc domain acts as a therapeuticagent by stimulating immune functions such as, e.g., antibody dependentcytotoxicity (ADC) or cell dependent cytotoxicity (CDC).

The general principle of display methodologies is to link a ligand(peptide, protein) to the gene coding for this ligand (see FIG. 2). Inthe phage display technique, this is obtained by fusing the ligand geneto the gene coding for a coat protein of a filamentous phage. Therecombinant phage genome is then introduced into Escherichia coli wherethe hybrid protein will be expressed together with all the other phageproteins. The fusion protein will then be incorporated into the phagecoat containing the phage genome (containing the ligand gene). Thesecreted phage particle displaying the ligand can be selected on animmobilized target while all the non-binding phages are washed away.After an elution step, the recovered phage is used to infect E. coli toallow the amplification of this phage for a new round of selection andeventually for the binding analysis.

Accordingly, a commercial peptide phage display library (12-mer peptidesin M13) was screened for peptides which bind to SPARC. The target, SPARCis an acidic glycoprotein with a PI of 4.6. By immobilization on96-wells plates with pH 9.6 coating buffer, Ph.D.-12 peptide library wasscreened four rounds to select peptide binders using phage displaytechnology. Specifically, bound phages were eluted with an acidiceluting solution in the 1st round of screening. Then the screeningstringency was enhanced gradually by decreasing the target proteinconcentration and increasing the percentage of Tween-20 in washingbuffer. At the same time, competitive elution with excess target wasadopted to improve the screening specificity. Finally, after four roundsof screening, ssDNA of selected clones were subjected to DNA sequencing.At the same time, the binding of the positive phages to the targetprotein was validated using phage ELISA.

The results of this screening of a peptide phage display library forSPARC binding peptides are shown in FIGS. 3 & 4. SPARC binding can bequantified by the number of phage clones isolated which encode peptideswith the same sequence (FIG. 3) or the avidity of SPARC binding asmeasured by the binding of peptide-expressing phage to SPARC-coatedmicrotiter plate wells (FIG. 4). Two of the peptides identified by phagedisplay, PD 15 (SEQ ID NO: 1) and PD 21 (SEQ ID NO: 2) were furthercharacterized.

PD 15 and PD 21 were then cloned into the expression vectorpFUSE-hIg1-Fc2 (FIG. 5), resulting in plasmids that encode PD 15-Fc andPD 21-Fc fusion proteins (FIG. 6). These fusion proteins were expressedand successfully purified as demonstrated by polyacrylamide gelelectrophoresis (FIG. 7).

Protein microarray analysis (see, FIG. 8) of PD 15 and PD 21 showed onlyminimal cross reactivity with the non-SPARC proteins in 5,000 proteinson the array assayed (Invitrogen, ProtoArray v.3).

Concentration dependent binding ELISA assays demonstrated PD 15 and PD21 binding to SPARC was shown to be only slightly weaker than that of ananti-SPARC antibody (FIG. 9). The SPARC binding Kd of PD 15 is4.1±0.6×10⁻⁸ M and that of PD 21 is 1.0±0.7×10⁻⁷ M. (The anti-SPARCantibody tested has a SPARC binding Kd of 6.2±3.4×10⁻⁹ M, i.e., theantibody binds SPARC only slightly more avidly.)

FIGS. 10 and 11 show the co-localization of SPARC (as indicated byimmunohistochemical (IHC) staining with an anti-SPARC antibody) and thebinding of PD 15 and PD 21 in sections of a human brain tumor (whichhave been epitope tagged for IHC staining.). As shown in FIG. 10, theliterature report of stabilin 1 binding to SPARC was not verified, asstab-Fc did not bind to tumor tissue whereas PD15 and PD21 did (FIG.11).

Sequence homology analysis of the SPARC-binding peptides isolated byphage display demonstrated that a number of the SPARC-binding peptidesequences isolated had sequence identities with a region of the Elastinshown in FIG. 12.

Thus, this Example demonstrates SPARC binding peptides can be identifiedby phage display and how to further characterize the identifiedpeptides. Of the two clones worked up, PD 15 and PD 21, PD 15 exhibiteda higher affinity for SPARC than PD 21 in ELSIA and IHC experiments.

EXAMPLE 2

The PD 15 and PD 21-Fc fusion proteins were assayed for antitumoractivity in a murine-human PC3 prostatecarcinoma xenograft model. BothPD 15 and PD 21-Fc fusion proteins demonstrated statisticallysignificant tumor growth inhibition (FIG. 13). PD15 exhibited betterantitumor activity than PD21 against the PC3 xenograft. In a mouse-humanHT29 colon xenograft model-PD21 exhibited better antitumor activity thanPD 15, with activity closely equivalent to Abraxane (FIG. 14).

EXAMPLE 3

This Example demonstrates the potential immungenicity of theSPARC-binding peptides.

ProPred is a graphical web tool for predicting MHC class II bindingregions in antigenic protein sequences (see Singh et al.: ProPred:prediction of HLA-DR binding sites. Bioinformatics 2001, 17(12):1236-7).The server implement matrix based prediction algorithm, employingamino-acid/position coefficient table deduced from literature. Thepredicted binders can be visualized either as peaks in graphicalinterface or as colored residues in HTML interface. This server might bea useful tool in locating the promiscuous binding regions that can bindto several HLA-DR alleles.

The results of a ProPred analysis of SPARC-binding peptides identifiedwith phage display, including PD 21 and PD 15 indicate that only a fewHLA-DR molecules will present these peptides and suggest that thepeptides will not be very immunogenic.

Any peptide disclosed herein, including, e.g., SEQ ID: 1-112 or 117,showing high affinity can be similarly analyzed for low or noimmunogenicity.

EXAMPLE 4

Antibody fragments also can be displayed on phages using differentformats. Single Chain Variable Fragment (scFv) is a fusion of thevariable regions of the heavy and light chains of immunoglobulins,linked together with a short (usually serine, glycine) linker. Thischimeric molecule retains the specificity of the originalimmunoglobulin, despite removal of the constant regions and theintroduction of a linker peptide. The most common formats for antibodyphage display include the use of scFv libraries. Large collections ofantibody variants can thus be screened for the presence of anantigen-binding clone.

The overall strategy was to first, screen a human antibody phage displaylibraries by ELISA with SPARC as antigen.

At the start, HuScL-3® was screened four rounds (three rounds withacidic elution and one round with competitive elution) and 17 positiveclones were selected by phage ELISA. DNA sequencing of these clonesrevealed two unique antibody sequences, between which the 1st sequencewas shared by 15 positive clones and the 2nd one was shared by remainingtwo positive clones. After that, the binding specificity of the twounique antibodies was validated by soluble scFv ELISA.

Next, HuScL-2® was screened for three rounds (two rounds withtrypsin-digestion elution and one round with competitive elution). Inthe end, 30 positive clones were selected by phage ELISA. According tothe sequencing results, 29 clones shared one antibody sequence and theremaining one clone encoded another unique antibody. After that, thebinding specificity of these two antibodies was validated by solublescFv ELISA as well.

Four unique ScFv against SPARC were identified, ScFv 3-1, ScFv 3-2, ScFv2-1, and ScFv 2-2 (SEQ ID NOs: 113-116).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of treating a mammal comprisingadministering to the mammal a therapeutically or diagnosticallyeffective amount of a pharmaceutical composition comprising atherapeutic or diagnostic agent coupled to a SPARC-binding-peptide (SBP)and a pharmaceutically acceptable carrier, wherein the SBP consists ofany one or more of SEQ ID NOS: 1-112 or
 117. 2. The method of claim 1,wherein the therapeutic or diagnostic agent is a diagnostic agentselected from the group consisting of radioactive agents, MRI contrastagents, X-ray contrast agents, ultrasound contrast agents, and PETcontrast agents.
 3. The method of claim 1, wherein the pharmaceuticalcomposition further comprises an albumin binding peptide (ABP)consisting of SEQ ID NO:
 120. 4. The method of claim 1, wherein themammal is a human patient.
 5. The method of claim 1, wherein the SBPconsists of SEQ ID NO:
 1. 6. The method of claim 1, wherein the SBPconsists of SEQ ID NO:
 2. 7. The method of claim 1, wherein thetherapeutic or diagnostic agent is a therapeutic agent selected from thegroup consisting of platinum compounds, antifolates, antimetabolites,antimitotics, DNA damaging agents, proapoptotics, differentiationinducing agents, antiangiogenic agents, antibiotics, tyrosine kinaseinhibitors, kinase inhibitors, biologically active agents, biologicalmolecules, hormones, peptides, antibodies, antibodies fragments andcombinations thereof.
 8. The method of claim 7, wherein the therapeuticagent is selected from the group consisting of radionuclides,adriamycin, ansamycin antibiotics, asparaginase, bleomycin, busulphan,cisplatin, carboplatin, carmustine, capecitabine, chlorambucil,cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin,daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide,epothilones, floxuridine, fludarabine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, mercaptopurine, meplhalan, methotrexate, rapamycin(sirolimus), mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel,pamidronate, pentostatin, plicamycin, procarbazine, rituximab,streptozocin, teniposide, thioguanine, thiotepa, taxanes, vinblastine,vincristine, vinorelbine, taxol, combretastatins, discodermolides,transplatinum, docetaxel, paclitaxel, taxanes, 5-fluorouracil,genistein, tTF, TNF, Smar1 derived p44 peptide, interferon, TRAIL, Smac,VHL, procaspase, caspase, and IL-2, a non-Fc domain antibody fragment,and combinations thereof.
 9. The method of claim 7, wherein thetherapeutic agent is an antibody fragment comprising a functionalantibody Fc domain.
 10. The method of claim 9, wherein the therapeuticagent is an antibody fragment which mediates one or more of complementactivation, cell mediated cytotoxicity, inducing apoptosis, inducingcell death, and opsinization.
 11. The method of claim 9, wherein thefunctional antibody Fc domain comprises SEQ ID NO: 118.